Abstract Relapse is the primary obstacle to cure in acute and chronic leukemia. The detection of measurable residual disease (MRD) is a direct measure of disease burden, treatment efficacy and is the strongest predictor of relapse. Although MRD has been established as a standard of care procedure and as a measurement of outcomes in clinical trials for chronic myeloid (CML) and acute lymphoblastic leukemia (ALL) it is more difficult to perform and standardize in acute myeloid leukemia (AML) and has not yet been integrated into trial design. Our objective in this proposal is to develop a single-cell DNA molecular diagnostic predictive for MRD in AML. Mission Bio has developed and commercially launched a novel microfluidic droplet platform, Tapestri, that performs high-throughput single-cell DNA sequencing. With a user friendly, cost effective and rapid workflow, Tapestri is capable of accurately genotyping 10,000 cells per run at hundreds of disease relevant loci. Through collaborations, we have used Tapestri to generate high-resolution maps of clonal architecture from longitudinally collected AML tumor samples and demonstrated the capability of identifying rare clones present of 0.1% of the tumor population. In this study, we will improve the Tapestri performance to allow detection of rare subclones present at 0.01% and use this platform to build an AML MRD-specific targeted sequencing panel. The Tapestri AML MRD panel will be deployed on up to 100 retrspective AML patient samples using paired diagnostic and remission samples and we will use the derived data to develop a relapse risk assessment using models that include single- cell genotyping data, intra-tumoral heterogeneity, and MRD status. Tapestri single-cell data will be benchmarked against existing technologies, including flow cytometry and bulk DNA sequencing. Having an ultra-sensitive MRD detection method capable of describing residual leukemic clones at the mutation level would be of great benefit by (1) allowing for more accurate prediction of AML relapse, (2) thus providing a platform for testing of MRD-directed intensification or de-escalation of therapy, (3) identifying actionable mutations present in residual leukemic clones that could serve as targets for therapy in the context of clinical trials, and (4) evaluating MRD status as a surrogate end point for new drug approvals.